Most cell separation methods provide enrichment of a cell population rather than true purification. Methods that provide pure cell preparations are often slow and have low recovery. A number of cell isolation/separation techniques have been employed previously for purifying/enriching or removing a cell population(s) from a suspension and can be divided into three categories (Kumar and Lykke, 1984, Cell separation: a review, Pathology. January; 16(1):53-62). First, methods that exploit physical differences between cell populations (cell density, cell size, cell charge, optical properties) such as isopycnic density gradient centrifugation, velocity sedimentation, electrophoresis, phase partitioning, and flow cytometry. Second, methods in which separation is based upon differences in properties of the cell surface (adhesion, and surface antigen such as affinity and magnetic methods). Third, methods that separate cells on the basis of their functional characteristics such as proliferation, phagocytosis, and antigen recognition.
Paradoxically, although the purpose is mainly to separate functionally different cell populations, there are very few existing methods that are actually based upon differences in cell function. Techniques based upon cell surface properties have a good correlation with functional differences between cell populations. In recent years, those methods have been more widely used, especially in the area of stem cell purification. A disadvantage with using affinity methods, for example, is that they can often be expensive or time-consuming to perform and can cause considerable damage to, or activation of, desired cells and/or can add undesirable agents to the purified or isolated cell suspensions (e.g toxins, proliferation-inducing agents, and/or antibodies). An additional problem in the purification of stem cells using antibody-based methods is that the most primitive stem cells may not possess the antibody-targeted cell surface marker (e.g CD34) and such cells will not be recovered.
The most widely used techniques are those that rely on physical differences and electrophoresis falls into this category. The main form of electrophoresis used up to now is free-flow electrophoresis. This form involves laminar flow of cells through a specially designed chamber within an electric field. The different mobilities (different charge to mass ratios) of the cells in the electric field allows the cells to separate and they are collected through multiple channels at the end of the chamber. The extent to which these correlate with functional properties of the cells is variable.
Cell electrophoresis is a high resolution separation method. In traditional electrophoresis, sub-populations of cells for which no affinity ligand has been developed and for which there is no distinct size or density range are often separable on the basis of their electrophoretic mobility, which may be related to their function. The electrophoretic mobility of a cell is directly correlated with the cellular negative surface charge density.
The surface charge on cells will vary depending on the cell type, relative freshness of the cells, and the pH of the electrophoresis buffer used for separation. At physiological pH (around neutral), cells have a net negative surface charge and when placed in an electric field, they are deflected or moved towards the anode. In electrophoresis devices reported in the literature, fractionation is based on electrophoretic mobilities of the cells (Smolka, Margel et al. 1979, Electrophoretic cell separation by means of microspheres, Biochim Biophys Acta. 1979 Dec. 3; 588(2):246-55).
Some of the challenges faced by early investigators of electrophoretic cell separation were excessive heat generation, degree of resolution of the separated fractions and scalability of the technology.
At present there is no acceptable and practical means to separated or select sperm populations other than the time-consuming, inefficient process of density gradient centrifugation.
The present inventors provide a reliable, reproducible, rapid, efficient, and cost-effective method of enriching a sperm population from semen or other solutions using membrane-based electrophoresis technology.